MicroRNAs (miRNAs) are 22-nt RNAs that pair to the mRNAs of plant and animal cells, primarily in their 3' untranslated regions (3'UTRs), to direct the posttranscriptional repression o these messages. More than 500 miRNA genes have been identified in humans, and because most human genes have been under selective pressure to maintain their pairing to miRNAs, it is no surprise that miRNAs have already been shown to play important roles in mammalian development and human disease, including viral infection and cancer. This proposal focuses on the regulatory targets and repressive effects of miRNAs, with the broad, long-term objective of understanding the roles of RNA in regulating gene expression. With recent advances in miRNA genomics, the ability to place miRNAs into gene regulatory networks is limited less by unknown miRNAs and more by unknown 3'UTRs, particularly the alternative 3'UTR isoforms found in some cells but not others. The specific aims of this proposal are: 1) to identify 3'UTRs of flies and vertebrates, 2) to identify additional mRNA and cellular features that influence miRNA targeting specificity, and 3) to determine the diversity and temporal sequence of miRNA repressive effects. Experiments of Aim #1 will use a newly developed high-throughput method both to characterize 3'UTRs of flies, zebrafish, and mammalian cell lines and to learn how 3'UTR isoforms change during development and cell-cycle arrest. They will also identify long noncoding RNAs in Caenorhabditis elegans, flies, and vertebrates. Experiments of Aim #2 will determine the degree to which poly(A)-tail length, mRNA stability and cellular features influence the efficacy of miRNA targeting. These results and those of Aim #1 will be used to provide a more precise and comprehensive list of predicted miRNA targets-a resource for all biologists, and of particular value for those concerned with eukaryotic gene regulation, development, and disease. Experiments of Aim #3 will determine the extent to which miRNA-mediated repression can be discerned from reduced transcript levels. They also will determine, on a global scale, the earliest detectable molecular consequences of targeting. Knowing which genes each miRNA represses and the molecular consequences of this repression will be useful for determining additional biological functions of miRNAs and learning how their dysfunction can contribute to disease.